WO2019183181A1 - Procédés d'amélioration de la persistance de lymphocytes t perfusés adoptivement - Google Patents

Procédés d'amélioration de la persistance de lymphocytes t perfusés adoptivement Download PDF

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WO2019183181A1
WO2019183181A1 PCT/US2019/023104 US2019023104W WO2019183181A1 WO 2019183181 A1 WO2019183181 A1 WO 2019183181A1 US 2019023104 W US2019023104 W US 2019023104W WO 2019183181 A1 WO2019183181 A1 WO 2019183181A1
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cells
days
hours
expanded
cancer
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Amir ALPERT
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Immatics US Inc
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Immatics US Inc
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Priority to CN201980027347.6A priority Critical patent/CN112512593A/zh
Priority to SG11202009082YA priority patent/SG11202009082YA/en
Priority to AU2019240039A priority patent/AU2019240039A1/en
Priority to JP2020549771A priority patent/JP7344898B2/ja
Priority to CA3094344A priority patent/CA3094344A1/fr
Priority to KR1020207030058A priority patent/KR20200133370A/ko
Priority to EA202092243A priority patent/EA202092243A1/ru
Priority to EP19772552.6A priority patent/EP3768330B1/fr
Publication of WO2019183181A1 publication Critical patent/WO2019183181A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A01N1/16—Physical preservation processes
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/86—Viral vectors
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
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    • C12N2501/20—Cytokines; Chemokines
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    • C12N2501/20—Cytokines; Chemokines
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/505—CD4; CD8
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2740/00—Reverse transcribing RNA viruses
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    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present disclosure provides for methods of improving the efficacy of T cells.
  • the disclosure further provides for methods of enhancing the persistence of T cells for adoptive cell transfer or therapy (ACT).
  • Cytokine sensitivity assays (CSA) and associated methodology capable of predicting the persistence of adoptively infused T Cells are further provided for by way of the instant disclosure.
  • the disclosure also provides for methods of treating cancer in a subject in need thereof as well as T cells populations produced by methods described herein.
  • Adoptive cell transfer or therapy is a form of immunotherapy that involves the ex vivo isolation and expansion of antigen-specific T cells for adoptive transfer back to patients.
  • ACT adoptive transfer or therapy
  • T cell persistence is considered to be a driving force for ACT efficacy, correlating T cell persistence/young phenotype to pre-clinical and clinical outcomes.
  • the common-gamma chain (yc)-cytokine IL-2 expands T cells.
  • High doses of IL-2 have also been used to expand ACT T cell cultures.
  • Enforced expression of IL-2 by T cells results in prolonged survival in vitro and maintains the tumor specificity and function.
  • IL-2 can promote differentiation of T cells, which may lead to an unfavorable phenotype for ACT usage.
  • yc-cytokines such as IL-7, IL-15, and IL-21 , have been described to play a role in memory T cell formation, proliferation, and survival, yet result in a lower degree of T cell differentiation but are still able to enhance anti-tumor responses.
  • U.S. 7,993,638 recites methods for treating a subject in need of treatment for cancer, including administering to the subject the activated cytotoxic T lymphocytes (CTLs); administering to the subject at least two cytokines including interferon-a-2b and interleukin- 2 (IL-2) that influence CTL persistence.
  • CTLs activated cytotoxic T lymphocytes
  • IL-2 interleukin- 2
  • U.S. 2015/0017120 recites methods of prolonging persistence of transferred cells, stimulating the proliferation of transferred cells, or stimulating a T cell-mediated immune response to a target cell population in a cancer subject receiving adoptive cell therapy (ACT), including: administering an extended-pharmacokinetic IL-2 to a cancer subject receiving ACT, in an amount effective to prolong the persistence of transferred cells in the subject.
  • ACT adoptive cell therapy
  • the disclosure provides for methods of improving the efficacy and viability of T cells.
  • the disclosure further provides for methods for producing T cells with improved efficacy for adoptive immunotherapy comprising
  • T cells from at least one healthy donor, patient, or individual, activating the T cells
  • the disclosure provides for methods for increasing the growth of T cells comprising
  • T cells from at least one healthy donor, patient, or individual,
  • the growth of the T cells expanded for about 3 to about 5 days is greater than that of activated T cells expanded for about 7 days or more after activation.
  • the disclosure provides for methods of decreasing cell death of T cells for use in adoptive immunotherapy comprising
  • T cells from at least one healthy donor, patient, or individual,
  • the disclosure further provides for methods wherein the activated T cells are expanded for about 4 days after activation and wherein the efficacy for adoptive
  • immunotherapy of the T cells is greater than that of activated T cells expanded for about 7 days or more after activation.
  • the disclosure further provides for methods wherein the activated T cells are expanded for about 3 days after activation and wherein the efficacy for adoptive
  • immunotherapy of the T cells is greater than that of activated T cells expanded for about 6 days or more after activation.
  • the disclosure further provides for methods for producing T cells with improved efficacy for adoptive immunotherapy comprising
  • T cells from at least one healthy donor, patient, or individual,
  • the disclosure provides for methods for producing T cells with improved efficacy for adoptive immunotherapy comprising
  • T cells from at least one healthy donor, patient, or individual,
  • the first period of time is from about 2 to about 5 days and said second period of time is from about 6 days to about 10 days; the first period of time is from about 3 to about 5 days and said second period of time is from about 7 days to about 10 days; the first period of time is from about 2 to about 5 days and said second period of time is from about 6 days to about 14 days; and the first period of time is less than about 6 days and said second period of time is greater than about 7 days.
  • the expanded T cells are CD4+ and/or CD8+ T cells.
  • the expanded T cells exhibit a naive T cells (TN) and/or stem memory T cells (Tscm)/T central memory (T C m) phenotype.
  • TN naive T cells
  • Tscm stem memory T cells
  • T C m T central memory
  • T cells are activated by a stimulator.
  • the stimulator comprises anti-CD3 antibody and an anti-CD28 antibody.
  • T cells described herein are used in adoptive immunotherapy in a patient in need of cancer treatment, wherein the cancer is selected from the group consisting of hepatocellular carcinoma (HCC), colorectal carcinoma (CRC), glioblastoma (GB), gastric cancer (GC), esophageal cancer, non-small cell lung cancer (NSCLC), pancreatic cancer (PC), renal cell carcinoma (RCC), benign prostate hyperplasia (BPH), prostate cancer (PCA), ovarian cancer (OC), melanoma, breast cancer, chronic
  • CLL lymphocytic leukemia
  • MCC Merkel cell carcinoma
  • SCLC small cell lung cancer
  • NHL Non-Hodgkin lymphoma
  • AML acute myeloid leukemia
  • GBC gallbladder cancer
  • UBC urinary bladder cancer
  • ALL acute lymphoblastic leukemia
  • UPC urinary bladder cancer
  • the disclosure provides for assays of evaluating T cells viability, comprising
  • T cells from at least one donor, patient, or individual,
  • the disclosure further provides for methods of producing T cells comprising obtaining T cells from at least one donor, patient, or individual, activating the T cells,
  • the T cells are obtained from at least one healthy donor, patient, or individual. In another aspect, the T cells are obtained from at least one cancer-free donor, patient, or individual.
  • the T cells are allogenic to the patient being treated. In another aspect, the T cells are autologous to the patient being treated.
  • the disclosure provides for freezing the expanded first portion of the activated T cells prior to culturing.
  • the disclosure provides for thawing the frozen expanded first portion of activated T cells prior to culturing.
  • the disclosure provides for resting the thawed expanded first portion of the activated T cells prior to culturing.
  • the disclosure provides for transducing activated T cells with a viral vector or a non-viral vector prior to expanding.
  • the vector may be a viral vector, such as a retroviral vector expressing a T cell receptor (TCR) or a lentiviral vector expressing a T cell receptor (TCR) or a non-viral vector, such as liposome, expressing a TCR.
  • a viral vector such as a retroviral vector expressing a T cell receptor (TCR) or a lentiviral vector expressing a T cell receptor (TCR) or a non-viral vector, such as liposome, expressing a TCR.
  • T cells expansion is measured over a period of time from about 1 day to about 15 days, from about 2 days to about 14 days, from about 3 days to about 13 days, from about 3 days to about 12 days, from about 3 days to about 1 1 days, from about 3 days to about 10 days, from about 3 days to about 9 days, from about 3 days to about 8 days, from about 3 days to about 7 days, from about 3 days to about 6 days, from about 3 days to about 5 days, from about 3 days to about 4 days, from about 4 days to about 6 days, or from about 4 days to about 5 days after activation.
  • the at least one cytokine is selected from the group consisting of (interleukin) IL-2, IL-7, IL-10, IL-12, IL-15, IL-21 , and a combination thereof.
  • the concentration of IL-2 is from about 10 U/ml to about 500 U/ml, from about 10 U/ml to about 450 U/ml, from about 10 U/ml to about 400 U/ml, from about 10 U/ml to about 350 U/ml, from about 10 U/ml to about 300 U/ml, from about 10 U/ml to about 250 U/ml, from about 10 U/ml to about 200 U/ml, from about 10 U/ml to about 150 U/ml, from about 10 U/ml to about 100 U/ml, from about 10 U/ml to about 50 U/ml, from about 20 U/ml to about 40 U/ml, from about 25 U/ml to about 35 U/ml, or from about 30 U/ml to about 35 U/ml.
  • the concentration of IL-7 provided herein is from 0.1 ng/ml to 50 ng/ml, from 0.1 ng/ml to 45 ng/ml, from 0.1 ng/ml to 40 ng/ml, from 0.1 ng/ml to 35 ng/ml, from 0.1 ng/ml to 30 ng/ml, from 0.1 ng/ml to 25 ng/ml, from 0.1 ng/ml to 20 ng/ml, from 0.1 ng/ml to 15 ng/ml, from 0.1 ng/ml to 10 ng/ml, from 0.1 ng/ml to 5 ng/ml, from 0.1 ng/ml to 4 ng/ml, from 0.1 ng/ml to 3 ng/ml, from 0.1 ng/ml to 2 ng/ml, from 0.1 ng/ml to 1 ng/ml, or from 0.1 ng/ml to 0.5
  • the concentration of IL-15 is from 0.1 ng/ml to 50 ng/ml, from 0.1 ng/ml to 45 ng/ml, from 0.1 ng/ml to 40 ng/ml, from 0.1 ng/ml to 35 ng/ml, from 0.1 ng/ml to 30 ng/ml, from 0.1 ng/ml to 25 ng/ml, from 0.1 ng/ml to 20 ng/ml, from 0.1 ng/ml to 15 ng/ml, from 0.1 ng/ml to 10 ng/ml, from 0.1 ng/ml to 5 ng/ml, from 0.1 ng/ml to 4 ng/ml, from 0.1 ng/ml to 3 ng/ml, from 0.1 ng/ml to 2 ng/ml, from 0.1 ng/ml to 1 ng/ml, or from 0.1 ng/ml to 0.5 ng/m
  • the disclosure further provides for methods wherein the cytokine response is selected from one or more of increased proliferation, reduced apoptosis, increased population of na ' ive T cells (TN) and/or stem memory T cells (Tscm)/T central memory (Tcm), and a combination thereof.
  • TN na ' ive T cells
  • Tscm stem memory T cells
  • Tcm T central memory
  • the resting step is carried out within a period of time from about 0.5 hour to about 48 hours, about 0.5 hour to about 36 hours, about 0.5 hour to about 24 hours, about 0.5 hour to about 18 hours, about 0.5 hour to about 12 hours, about 0.5 hour to about 6 hours, about 1 hour to about 6 hours, about 2 hours to about 5 hours, about 3 hours to about 5 hours, or about 1 hours to about 24 hours, about 2 to about 24 hours, about 12 to about 48 hours, about 0.5 hour to about 120 hours, about 0.5 hour to about 108 hours, about 0.5 hour to about 96 hours, about 0.5 hour to about 84 hours, about 0.5 hour to about 72 hours, or about 0.5 hour to about 60 hours.
  • the anti-CD3 antibody and the anti- CD28 antibody each have a concentration of from about 0.1 pg/ml to about 10.0 pg/ml, about 0.1 pg/ml to about 8.0 pg/ml, about 0.1 pg/ml to about 6.0 pg/ml, about 0.1 pg/ml to about 4.0 pg/ml, about 0.1 pg/ml to about 2.0 pg/ml, about 0.1 pg/ml to about 1.0 pg/ml, about 0.1 pg/ml to about 0.8 pg/ml, about 0.1 pg/ml to about 0.6 pg/ml, about 0.1 pg/ml to about 0.5 pg/ml, about 0.1 pg/ml to about 0.25 pg/ml, about 0.2 pg/ml to about 0.5 pg/ml, about 0.2 pg/ml to about 0.5 p
  • the activation is carried out within a period of from about 1 hour to about 120 hours, about 1 hour to about 108 hours, about 1 hour to about 96 hours, about 1 hour to about 84 hours, about 1 hour to about 72 hours, about 1 hour to about 60 hours, about 1 hour to about 48 hours, about 1 hour to about 36 hours, about 1 hour to about 24 hours, about 2 hours to about 24 hours, about 4 hours to about 24 hours, about 6 hours to about 24 hours, about 8 hours to about 24 hours, about 10 hours to about 24 hours, about 12 hours to about 24 hours, about 12 hours to about 72 hours, about 24 hours to about 72 hours, about 6 hours to about 48 hours, about 24 hours to about 48 hours, about 6 hours to about 72 hours, or about 1 hours to about 12 hours.
  • T cells obtained by methods described herein are CD3 + CD8 + T cells.
  • the disclosure provides for methods of assessing viability of T cells by utilizing methods and method steps described herein.
  • methods described herein only include in vitro method steps.
  • methods described herein do not include in vivo method steps.
  • methods described herein include a combination of method steps performed in vitro and in vivo.
  • methods described herein do not include analysis or evaluation by utilizing transgenic animals, for example, transgenic mice.
  • methods described herein are capable of determining conditions for T cells production and/or T cell viability faster than methods involving utilizing a transgenic animal, for example, a transgenic mouse.
  • methods described herein provide for viable T cells capable of being utilized for infusion into a patient or subject in need thereof.
  • methods described herein are performed in vitro and are predicative of in vivo results.
  • the disclosure provides for high throughput in vitro assays that are predictive of the in vivo viability of T cells for transfusion.
  • the description provides for cytokine response (CR) assays and associated methodology capable of predicting the persistence of adoptively infused T cells.
  • the description provides for cytokine sensitivity assays that are capable of measuring the effect of in vitro expansion length on ability to respond to cytokine and survive in the absence of continual cytokine stimulation
  • methods described herein may be used to determine which types of T cells persist in vivo by utilizing high-throughput in vitro methodology.
  • compositions comprising T cells produced and described herein are further provided for.
  • pharmaceutical compositions described herein include a pharmaceutically acceptable carrier, excipient, or salt thereof.
  • T cell population produced by methods described herein are further provided for by way of the disclosure.
  • the T cells are engineered T cells.
  • the description provides for methods for predicting in vivo persistence of T cells in a solid tumor, comprising
  • thawing cryopreserved T cells expanded for a plurality of expansion times, resting the thawed T cells in the absence of a cytokine
  • cytokines are added to the culture
  • the added one or more cytokines are depleted, sampling the cultured T cells at a plurality of time points during the at least one cycle of time,
  • the plurality of expansion times are from about 1 day to about 15 days, from about 2 days to about 14 days, from about 3 days to about 13 days, from about 3 days to about 12 days, from about 3 days to about 1 1 days, from about 3 days to about 10 days, from about 3 days to about 9 days, from about 3 days to about 8 days, from about 3 days to about 7 days, from about 3 days to about 6 days, from about 3 days to about 5 days, from about 3 days to about 4 days, from about 4 days to about 6 days, or from about 4 days to about 5 days after activation.
  • the one cycle of time is 1 -10 days per cycle, 2-10 days per cycle, 3-10 days per cycle, 4-10 days per cycle, 5-10 days per cycle, 6-10 days per cycle, 7-10 days per cycle, 8-10 days per cycle, or 9-10 days per cycle,
  • the at least one cycle of time is 1 cycle of time, 2 cycles of time, 3 cycles of time, 4 cycles of time, 5 cycles of time, 6 cycles of time, 7 cycles of time, 8 cycles of time, 9 cycles of time, or 10 cycles of time.
  • the solid tumor is selected from the group consisting of hepatocellular carcinoma (HCC), colorectal carcinoma (CRC), glioblastoma (GB), gastric cancer (GC), esophageal cancer, non-small cell lung cancer (NSCLC), pancreatic cancer (PC), renal cell carcinoma (RCC), benign prostate hyperplasia (BPH), prostate cancer (PCA), ovarian cancer (OC), melanoma, breast cancer, Merkel cell carcinoma (MCC), small cell lung cancer (SCLC), gallbladder cancer and cholangiocarcinoma (GBC, CCC), urinary bladder cancer (UBC), and uterine cancer (UEC).
  • HCC hepatocellular carcinoma
  • CRCC colorectal carcinoma
  • GB glioblastoma
  • GC gastric cancer
  • esophageal cancer non-small cell lung cancer
  • NSCLC pancreatic cancer
  • PC renal cell carcinoma
  • BPH benign prostate hyperplasia
  • PCA prostate cancer
  • OC ova
  • FIG. 1 shows T cell apoptosis (e.g., re-stimulation induced cell death (RICD) and cytokine withdrawal induced cell death (CWID) and memory formation.
  • RCD re-stimulation induced cell death
  • CWID cytokine withdrawal induced cell death
  • FIG. 2 shows model of in vivo T cell survival in ACT targeting liquid tumors and solid tumors by inhibiting intrinsic or extrinsic apoptotic pathway, respectively.
  • FIG. 3 shows model of testing in vivo T cell survival in ACT targeting liquid tumors and solid tumors by serial killing assay or cytokine sensitivity assay, respectively.
  • FIG. 4 shows cytokine sensitivity assay according to one embodiment of the present disclosure.
  • FIG. 5A-5D show T SCm -like formation during in vitro expansion characterized by CD45RO(low) and CCR7+.
  • FIG. 6A-6I show early expanded T SCm retain IL-15 cytokine sensitivity across 21 Days in assay.
  • FIG. 7A-7C show that early expanded cells (expansion for about 4 days) demonstrate increased cell growth relative to expansion at 7 and 10 days.
  • the label under the graphs represents the amount of cytokine used.
  • a linear quadratic line fit is used to model cell behaviour. T-cells expanded for 4, 7, or 10 days were assessed via in the presence of 10 ng/ml IL-7 (A), 10 ng/ml IL-15 (B), or 300 U/mL IL-2 (C) over a period of 21 days with sampling every 2-3 days. Fold growth is calculated as the ratio of the starting T- cell number to the T-cell number at the designated time point. Note that each plot has a different scale on the Y-axis to facilitate data visualization. Best fit lines are derived by linear quadratic equations of cell survival.
  • FIGS. 8A-8C show shortened in vitro expansion of T cells (expansion for about 4 days) correlates with increased survival at higher cytokine concentrations relative to expansion at 7 and 10 days.
  • T-cells expanded for 4, 7, or 10 days were assessed via in the presence of 300 U/ml IL-2 (A), 10 ng/ml IL-7 (B), 10 ng/ml IL-15 (C), or over a period of 21 days with sampling every 2-3 days.
  • Integrated survival is the area under the curve of the fold growth plots as shown in FIGS. 7A-7C. Each point represents three technical replicates of each donor with a total of 3 donors shown.
  • FIGS. 8D-8F show shortened in vitro expansion of transduced T cells correlates with increased survival at higher cytokine concentrations.
  • FIGS. 9A-9C show shortened in vitro expansion of T cells correlates with increased survival at lower cytokine concentrations.
  • FIG. 10A-10C show shortened in vitro expansion of T cells correlates with reduced apoptosis.
  • FIGS. 11 A-1 1 C show shortened in vitro expansion of T cells correlates with reduced apoptosis at higher cytokine concentrations.
  • T-cells expanded for 4, 7, or 10 days were assessed via in the presence of 300 U/ml IL-2 (A), 10 ng/ml IL-7 (B), or 10 ng/ml IL-15 (C), over a period of 21 days with sampling every 2-3 days.
  • Integrated apoptosis is calculated based on the percentage of lymphocytes staining positive for propidium iodide and annexin-V by day 10 in the assay. Each point represents three technical replicates of each donor with a total of 3 donors shown.
  • FIG. 12A-12C show shortened in vitro expansion of T cells correlates with reduced apoptosis.
  • FIG. 13A-13C show shortened in vitro expansion of T cells correlates with increased cell division in the presence of (A) IL-7, (B) IL-15, and (C) IL-2.
  • FIGS. 14A-14C show shortened in vitro expansion of transduced T cells correlates with increased cell division at higher cytokine concentrations.
  • T-cells expanded for 4, 7, or 10 days were assessed via in the presence of 300 U/ml IL-2 (A), 10 ng/ml IL-7 (B), 10 ng/ml IL-15 (C), or over a period of 21 days with sampling every 2-3 days.
  • FIG. 15A-15C show shortened in vitro expansion of T cells correlates with increased sensitivity to (A) IL-7, (B) IL-15, and (C) IL-2.
  • FIGS. 16A-16C show shortened in vitro expansion of transduced T cells correlates with increased cell division at higher cytokine concentrations.
  • FIG. 16D shows shortened in vitro expansion of transduced T cells correlates with increased CD25 expression.
  • FIG. 17 shows correlation between IL-2 receptor (CD25) expression and survival/division in the presence of IL-2.
  • FIG. 18 shows correlation between IL-15 receptor (CD122) expression and survival/division in the presence of IL-15.
  • FIG. 19 shows correlation between IL-7 receptor (CD127) expression and survival/division in the presence of IL-7.
  • FIG. 20 shows shortened in vitro expansion of T cells retain T cell potentials.
  • FIGS. 21 A shows cell memory compartments were measured by flow cytometry at day 0 and every 7 days during the 21 -day culturing period.
  • Tnaive/scm CCR7+CD45RO-
  • Tcm CCR7+CD45RO+
  • Tem CCR7-CD45RO+
  • T eff CCR7-CD45RO-.
  • FIG. 21 B shows input cells were labeled with PkH proliferation dye at culture initiation and the proliferation of distinct memory compartment were measured based on PkH dilution by day 7 in culture period.
  • FIG. 22 shows continual loss of telomere length during CD3/CD28 T-cell expansion.
  • the relative telomere length was assessed by fluorescence in situ hybridization relative to a tumor cell line control in 4 healthy donors (D1 -D4).
  • Each sample point represents a replicate of a technical duplicate.
  • FIG. 23 shows reduced telomerase activity with prolonged CD3/CD28 T-cell expansion. Telomerase activity was measured via an ELISA based colorimetric assay from whole cell lysate of cells taken from day 4, 7, or 10 in T-cell expansion. Each point represents a technical triplicate sample from a total of 5 biological replicates.
  • FIG. 24 shows T-cell differentiation during CD3/CD28 Manufacturing from three biological donors D4, D5, and D6.
  • Representative PBMCs were cultured and then phenotyped by flow cytometry at the indicated expansion day.
  • FIG. 25 shows loss of costimulation during CD3/CD28 manufacturing from three biological donors D1 , D7, and D8. CD27 and CD28 expression was assessed via flow cytometry on day 4, 7, and 10 during the T-cell expansion period.
  • FIG. 26 shows differential gene expression analysis identifying clusters of the earlier expanded cells as a unique cluster compared to later expanded cells.
  • Three biological donors D4, D5, and D6 were expanded for 4, 7, or 10 days and then whole RNA was isolated and sent to Novogene for RNA sequencing analysis and bioinformatics.
  • FIG. 27 shows RNAseq analysis during T-cell manufacturing. Volcano plot representation of RNAseq data during T-cell manufacturing comparing (A) day 4 vs day 7, (B) day 4 vs day 10, and (C) day 7 vs day 10. DEGs cut-off was set to 1 -fold up or down with a padj-value of less than 0.05. Number of DEGs is shown in the key for each plot.
  • FIG. 28 shows Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis during T-cell manufacturing.
  • the left panels show the pathways that are
  • day_7 vs day_4_down indicates pathways that were down regulated in the day 7 sample vs the day 4 sample).
  • minimally expanded engineered T cells demonstrate greater clinical efficacy as compared to T cells expanded for extended in vitro periods due to an increased naivety and ability to proliferate and persist in-vivo.
  • the minimally expanded engineered T cells are expanded for about 3 to about 5 days relative to extended expression of about 7 to about 10 days.
  • T cells with a shorter expansion time of about 3 to about 5 days exhibit an increased cytokine response by 1 ) proliferation, 2) reduced apoptosis, and 3) persistence over T cells produced by the same method but with an increased expansion time of about 7 to about 10 days.
  • adoptive cell transfer or therapy comprises a treatment method, in which cells are removed from a donor, cultured and/or manipulated in vitro, and administered to a patient for the treatment of a disease.
  • transferred cells may be autologous cells, meaning that the patient acts as his or her own donor.
  • transferred cells may be lymphocytes, e.g., T cells.
  • transferred cells may be genetically engineered prior to administration to a patient.
  • the transferred cells can be engineered to express a T cell receptor (TCR) having specificity for an antigen of interest.
  • transferred cells may be engineered to express a chimeric antigen receptor (CAR).
  • transferred cells may be engineered (e.g., by transfection or conjugation) to express a molecule that enhances the anti-tumor activity of the cells, such as a cytokine (IL-2, IL-12), an anti-apoptotic molecule (BCL-2, BCL-X), or a chemokine (CXCR2, CCR4, CCR2B).
  • transferred cells may be engineered to express both a CAR and a molecule that enhances anti-tumor activity or persistence of cells.
  • the disclosure relates to methods wherein the outcome of Adoptive cell transfer or therapy (ACT) can be improved by administering minimally expanded T cells to cancer subjects.
  • ACT Adoptive cell transfer or therapy
  • expanded engineered T cells described herein are useful for treating a disorder associated with abnormal apoptosis or a differentiative process (e.g., cellular proliferative disorders or cellular differentiative disorders, such as cancer).
  • a disorder associated with abnormal apoptosis or a differentiative process e.g., cellular proliferative disorders or cellular differentiative disorders, such as cancer.
  • a differentiative process e.g., cellular proliferative disorders or cellular differentiative disorders, such as cancer.
  • Examples of cellular proliferative and/or differentiative disorders may include cancer (e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias).
  • a metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver.
  • compositions of the present disclosure can be administered to a patient who has cancer.
  • cancer or“cancerous”
  • hyperproliferative or “neoplastic”
  • neoplastic may be used to refer to cells having the capacity for autonomous growth (i.e., an abnormal state or condition characterized by rapidly proliferating cell growth).
  • Hyperproliferative and neoplastic disease states may be categorized as pathologic (i.e., characterizing or constituting a disease state), or they may be categorized as non- pathologic (i.e., as a deviation from normal but not associated with a disease state).
  • pathologic i.e., characterizing or constituting a disease state
  • non- pathologic i.e., as a deviation from normal but not associated with a disease state
  • the terms are meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.“Pathologic hyperproliferative” cells may occur in disease states characterized by malignant tumor growth. Examples of non- pathologic hyperproliferative cells may include proliferation of cells associated with wound repair.
  • cancer or“neoplasm” may be used to refer to malignancies of the various organ systems, including those affecting the lung, breast, thyroid, lymph glands and lymphoid tissue, gastrointestinal organs, and the genitourinary tract, as well as to
  • adenocarcinomas which may be generally considered to include malignancies, such as most colon cancers, renal cell carcinoma, prostate cancer and/or testicular tumors, non small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
  • the cancer can be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyo sarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, cervical cancer, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, peritoneum, omentum, and mesentery cancer
  • carcinoma refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas.
  • Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary.
  • the term may also include carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues.
  • An“adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
  • hematopoietic neoplastic disorders may include diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.
  • the diseases may arise from poorly differentiated acute leukemias (e.g., erythroblastic leukemia and acute megakaryoblastic leukemia).
  • Additional exemplary myeloid disorders may include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991 ) Crit. Rev. in Oncol./Hemotol. 1 1 :267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • PLL prolymphocytic leukemia
  • HLL hairy cell leukemia
  • malignant lymphomas may include but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease.
  • ATL adult T cell leukemia/lymphoma
  • CCL cutaneous T cell lymphoma
  • LGF large granular lymphocytic leukemia
  • Hodgkin's disease Reed-Sternberg disease.
  • therapeutically effective amount may vary not only on the particular compositions selected, but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will ultimately be at the discretion of the patient's physician or pharmacist.
  • the length of time during which minimally expanded engineered T cells used in the instant methods may be given varies on an individual basis. It will be appreciated by those skilled in the art that reference herein to treatment extends to prophylaxis as well as the treatment of the noted cancers and symptoms.
  • T cell or“T lymphocyte” may include thymocytes, naive T
  • T cells immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes.
  • Illustrative populations of T cells suitable for use in particular embodiments include, but are not limited to, helper T cells (HTL; CD4+ T cell), a cytotoxic T cell (CTL; CD8+ T cell), CD4+CD8+ T cell, CD4-CD8- T cell, or any other subset of T cells.
  • helper T cells HTL; CD4+ T cell
  • CTL cytotoxic T cell
  • CD4+CD8+ T cell CD4-CD8- T cell, or any other subset of T cells.
  • T cells suitable for use in particular embodiments include, but are not limited to, T cells expressing one or more of the following markers: CD3, CD4, CD8, CD27, CD28, CD45RA, CD45RO, CD62L, CD127, CD197, and HLA-DR and if desired, can be further isolated by positive or negative selection techniques.
  • a peripheral blood mononuclear cell refers to any blood cell with a round nucleus (i.e., a lymphocyte, a monocyte, or a macrophage). These blood cells are a critical component in the immune system to fight infection and adapt to intruders.
  • the lymphocyte population consists of CD4+ and CD8+ T cells, B cells and Natural Killer cells, CD14+ monocytes, and basophils/neutrophils/eosinophils/dendritic cells. These cells are often separated from whole blood or from leukopacks using FICOLLTM, a hydrophilic
  • PBMCs refers to a population of cells comprising at least T cells, and optionally NK cells, and antigen presenting cells.
  • activation refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. In particular embodiments, activation can also be associated with induced cytokine production, and detectable effector functions.
  • activated T cells refers to, among other things, T cells that are proliferating. Signals generated through the TCR alone are insufficient for full activation of the T cell and one or more secondary or costimulatory signals are also required. Thus, T cell activation comprises a primary stimulation signal through the TCR/CD3 complex and one or more secondary costimulatory signals. Costimulation can be evidenced by proliferation and/or cytokine production by T cells that have received a primary activation signal, such as stimulation through the CD3/TCR complex or through CD2.
  • a resting T cell means a T cell that is not dividing or producing cytokines. Resting T cells are small (approximately 6-8 microns) in size compared to activated T cells (approximately 12-15 microns).
  • a primed T cell is a resting T cell that has been previously activated at least once and has been removed from the activation stimulus for at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, at least about 108 hours, or at least about 120 hours.
  • resting may be carried out within a period of from about 0.5 hour to about 120 hours, about 0.5 hour to about 108 hours, about 0.5 hour to about 96 hours, about 0.5 hour to about 84 hours, about 0.5 hour to about 72 hours, about 0.5 hour to about 60 hours, about 0.5 hour to about 48 hours, about 0.5 hour to about 36 hours, about 0.5 hour to about 24 hours, about 0.5 hour to about 18 hours, about 0.5 hour to about 12 hours, about 0.5 hour to about 6 hours, about 1 hour to about 6 hours, about 2 hours to about 5 hours, about 3 hours to about 5 hours, or about 4 hours to about 5 hours.
  • Primed T cells usually have a memory phenotype.
  • Embodiments of the present disclosure may include resting in the absence of cytokines or in the presence of cytokines, e.g., IL-2, IL-7, IL-10, IL-12, IL-15, IL-21 , or a combination thereof, such as IL-7 + IL-15, for from about 0.5 hour to about 48 hours, about 0.5 hour to about 36 hours, about 0.5 hour to about 24 hours, about 0.5 hour to about 18 hours, about 0.5 hour to about 12 hours, about 0.5 hour to about 6 hours, about 1 hour to about 6 hours, about 2 hours to about 5 hours, about 3 hours to about 5 hours, about 4 hours to 6 hours, about 1 hours to about 24 hours, about 2 to about 24 hours, about 12 to about 48 hours, about 0.5 hour to about 120 hours, about 0.5 hour to about 108 hours, about 0.5 hour to about 96 hours, about 0.5 hour to about 84 hours, about 0.5 hour to about 72 hours, or about 0.5 hour to about 60 hours, e.g., about
  • Controlled expansion and contraction of lymphocytes both during and after an adaptive immune response may be imperative to sustaining a healthy immune system.
  • lymphocyte apoptosis Both extrinsic and intrinsic pathways of lymphocyte apoptosis may be programmed to eliminate cells at the proper time to ensure immune homeostasis. Without this lymphocyte apoptosis barrier, prolonged persistence and/or unchecked accumulation of activated lymphocytes can result in immunopathology, autoimmunity, and lymphoid cancers.
  • FIG. 1 shows, like most somatic cells, naive and memory T cells may operate in a generally quiescent metabolic state and utilize mitochondrial oxidative phosphorylation (OXPHOS) for ATP generation. Following T cell receptor (TCR) stimulation, however, responding T cells rapidly switch to using glycolysis even in the presence of oxygen
  • OXPHOS mitochondrial oxidative phosphorylation
  • Activated T cells may proliferate and acquire potent effector functions (e.g. IFN-g production), which may be linked to glycolytic metabolism. These changes in cellular metabolism over the course of a T cell response may profoundly influence cell survival and differentiation, including the generation of memory. During this window of expansion and aerobic glycolysis, however, effector T cells may become sensitive to restimulation-induced cell death (RICD).
  • Restimulation induced cell death is an apoptotic program that may ultimately set an upper limit for effector T cell expansion during an infection.
  • RICD sensitivity may be dependent on prior activation, cell cycle induction via cytokines, such as IL-2, and a subsequent, strong restimulation signal propagated through the TCR, which induces apoptosis in a subset of effectors.
  • cytokines such as IL-2
  • a subsequent, strong restimulation signal propagated through the TCR which induces apoptosis in a subset of effectors.
  • naive and resting memory T cells may be relatively resistant to RICD.
  • this self-regulatory death pathway may help maintain immune homeostasis by precluding excessive, non-specific immunopathological damage to the host.
  • a defect in RICD contributes to excessive T cell accumulation and lethal damage to host tissues, as noted in patients with X-linked lymphoproliferative disorder.
  • Cytokine withdrawal-induced cell death is an apoptosis program responsible for culling the majority of effector T cells, triggered by waning cytokines, e.g., IL-2, levels after an infection is cleared and may save a select few that survive as memory T cells. While excessive anabolic metabolism (e.g., glycolysis) may leave effector T cells more susceptible to RICD, catabolic metabolism (e.g., autophagy and fatty acid oxidation (FAO)), on the other hand, can protect T cells derived from distinct memory compartments from death induced by cytokine withdrawal. CWID sensitivity, therefore, may play a major role in determining which and how many T cells survive contraction and enter the memory pool, influencing secondary responses derived from distinct memory subsets.
  • cytokines e.g., IL-2
  • CWID and RICD may operate at different phases of the immune response as hard-wired feedback response programs, influenced by the dynamic localization of cells, antigen, and cytokine. Both processes are somewhatly regulated by the availability of antigen and IL-2 as well as other growth/survival cytokines. Mechanistically, these two processes may eliminate T cells through distinct biochemical mechanisms of apoptosis, known as the intrinsic and extrinsic pathways. The intrinsic pathway is controlled by relative expression of Bcl-2 family proteins that regulate mitochondrial outer membrane potential (MOMP). When mitochondria are depolarized, cytochrome c release catalyzes the cleavage and activation of procaspase 9. Extrinsic apoptosis is signaled principally through death receptors (DRs) of the tumor necrosis factor receptor (TNFR) superfamily, such as Fas.
  • DRs death receptors
  • TNFR tumor necrosis factor receptor
  • CWID induces intrinsic apoptosis.
  • Withdrawal of IL-2 or other g-chain cytokines specifically upregulates and activates Bim, a key pro-apoptotic protein that antagonizes the function of anti-apoptotic Bcl-2 family proteins (e.g. Bcl-2, Bcl-xL, and Mcl-1 ) and activates Bax, which causes mitochondrial permeabilization.
  • Bcl-2, Bcl-xL, and Mcl-1 a key pro-apoptotic protein that antagonizes the function of anti-apoptotic Bcl-2 family proteins (e.g. Bcl-2, Bcl-xL, and Mcl-1 ) and activates Bax, which causes mitochondrial permeabilization.
  • RICD may be attributed to an extrinsic apoptosis signal through Fas, which may be stimulated in cis or in trans by membrane- anchored FasL exposed on the surface of restimulated T
  • one objective of ex vivo T cell expansion may be to increase the amount of memory forming cells, such as na ' ive T cells (TN) and/or stem memory T cells (Tscm)/T central memory (T C m).
  • TN na ' ive T cells
  • Tscm stem memory T cells
  • T C m T central memory
  • FIG. 2 shows differences of conventional ACT T cells for treating solid tumors and liquid tumors.
  • T cells may be activated by anti-CD3 and anti- CD28 antibodies, followed by expansion for a period of time.
  • Activated/expanded engineered T cells in a solid tumor environment with reduced access of cognate antigen compared to liquid tumors, non-cognate antigens, and limited apoptosis inhibitors may undergo intrinsic apoptotic pathways, e.g., damage induced cell death (DICD) or CWID, induced during ex vivo expansion.
  • DICD damage induced cell death
  • CWID damage induced cell death
  • activated/expanded engineered T cells in liquid tumor environment with cognate antigen-rich environment with tumors and antigen presenting cells may be less likely to undergo apoptosis from CWID, but may be more likely to undergo activation induced cell death (AICD) from increase antigen stimulation, indicating that treatment of solid tumors may require T cells to withstand CWID more than AICD.
  • AICD activation induced cell death
  • FIG. 3 shows, to test the ability of in vitro expanded T cells to survive cytokine stimulation withdrawal, e.g., in solid tumors, cytokine sensitivity assays may be used.
  • cytokine sensitivity assays may be used.
  • serial killing assays may be used.
  • Table 1 summarize differences of T cell survival in vivo between liquid tumors and solid tumors.
  • in vitro expanded T cells in ACT targeting solid tumors in antigen deprived environments may be more dependent on cytokines for survival than those targeting liquid tumors
  • in vitro memory formation and CWID reduction may be more critical for in vitro expanded T cells targeting solid tumors than those targeting liquid tumors.
  • T cell types that could persist in vivo in a high-throughput patient specific fashion for ACT may increase clinical efficacy of targeting solid tumors.
  • Cytokine sensitivity assays of the present disclosure may be used to predict and select which types of expanded T cells that could persist in vivo in antigen deprived environments.
  • a source of T cells may be obtained from a subject.
  • T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • any number of T cell lines available in the art may be used.
  • T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FicollTM separation.
  • cells from the circulating blood of an individual may be obtained by apheresis.
  • the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
  • the cells may be washed with phosphate buffered saline (PBS), or with a wash solution that lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium can lead to magnified activation.
  • PBS phosphate buffered saline
  • washing step may be accomplished by methods known to those in the art, such as by using a semi-automated“flow-through” centrifuge (for example, the Cobe 2991 ceil processor, the Baxter CytoMate, or the
  • the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca 3+ - free, Mg 2+ -free PBS, PlasmaLyte A, or other saline solution with or without buffer.
  • biocompatible buffers such as, for example, Ca 3+ - free, Mg 2+ -free PBS, PlasmaLyte A, or other saline solution with or without buffer.
  • the undesirable components of the apheresis sample may be removed, and the cells directly resuspended in culture media.
  • T cells may be isolated from peripheral blood
  • T cells by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient or by counterflow centrifugal elutriation.
  • a specific subpopulation of T cells can be further isolated by positive or negative selection techniques.
  • T cells may be isolated by incubation with anti-CD3/anti- CD28 (i.e., 3x28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells.
  • Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • One method may be cell sorting and/or selection via negative magnetic immune- adherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
  • a monoclonal antibody cocktail typically may include antibodies to CD14, CD20, CD1 1 b, CD16, FILA-DR, and CD8.
  • it may be desirable to enrich for or positively select for regulatory T cells which typically may express CD4+, CD25+, CD62L1 , GITR+, and FoxP3+.
  • T regulatory cells may be depleted by anti-CD25 conjugated beads or other similar method of selection.
  • the concentration of cells and surface can be varied.
  • a concentration of 2 billion cells/ml may be used.
  • a concentration of 1 billion cells/ml may be used.
  • greater than 100 million cells/ml may be used.
  • a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml may be used.
  • a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml may be used.
  • concentrations of 125 or 150 million cells/ml can be used.
  • Using high concentrations can result in increased cell yield, cell activation, and cell expansion.
  • use of high cell concentrations may allow more efficient capture of cells that may weakly express target antigens of interest, such as CD28- negative T cells, or from samples where there are many tumor cells present (i.e., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain.
  • using high concentration of cells may allow more efficient selection of CD8+ T cells that normally have weaker CD28 expression.
  • concentrations of cells By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells may be minimized. This may select for cells that express high amounts of desired antigens to be bound to the particles.
  • the cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681 ;
  • the autologous cells can be administered by any suitable route as known in the art.
  • the cells may be administered as an intra-arterial or intravenous infusion, which lasts about 30 to about 60 minutes.
  • Other exemplary routes of administration may include intraperitoneal, intrathecal and intralymphatic.
  • any suitable dose of autologous cells can be administered.
  • from about 1.0x10 8 cells to about 1.0x10 12 cells may be administered.
  • from about 1.0x10 10 cells to about 13.7x10 10 T-cells may be administered, with an average of around 5.0x10 10 T-cells.
  • from about 1.2x10 10 to about 4.3x10 10 T-cells may be administered.
  • the autologous cells used for ACT may be lymphocytes, e.g., T cells.
  • the T cells may be“young” T cells, e.g., between 19-35 days old, as described in, for example, U.S. Pat. No. 8,383,099, incorporated by reference herein in its entirety. Young T cells are believed to have longer telomeres than older T cells, and longer telomere length may be associated with improved clinical outcome following ACT in some instances.
  • T cells and methods of producing T cells described herein may be used in conjunction with one or more of representative strategies for ACT: tumor infiltrating lymphocytes (TIL), antigen-expanded CD8+ and/or CD4+ T cells, T cells genetically modified to express a T cell receptor (TCR) that specifically recognizes a tumor antigen, and T cells genetically modified to express a chimeric antigen receptor (CAR).
  • TIL tumor infiltrating lymphocytes
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • TIL Tumor Infiltrating Lymphocytes
  • One ACT strategy involves the transplantation of autologous TIL expanded ex wVo from tumor fragments or single cell enzymatic digests of tumor metastases. T cell infiltrates in tumors are polyclonal in nature and collectively recognize multiple tumor antigens. See, for example, Rosenberg et al., N. Engl. J. Med. (1988) 319:1676-1680, which is herein incorporated by reference in its entirety.
  • tumors may be resected from patients and cut into small (for example, 3-5 mm 2 ) fragments under sterile conditions.
  • the fragments may be placed into culture plates or flasks with growth medium and treated with high-dose IL-2.
  • This initial TIL expansion-phase also known as the“Pre-REP” phase
  • This initial TIL expansion-phase typically lasts about 3 to about 5 weeks, during which time about 5x10 7 or more TILs may be produced.
  • the resulting TILs may be then further expanded (e.g., following a rapid expansion protocol (REP)) to produce TILs suitable for infusion into a subject.
  • the pre-REP TILs can be cryopreserved for later expansion, or they may be expanded immediately.
  • Pre-REP TILs can also be screened to identify cultures with high anti-tumor reactivity prior to expansion.
  • a typical REP may involve activating TILs using a T-cell stimulating antibody, e.g., an anti- CD3 mAb, in the presence of irradiated PBMC feeder cells.
  • the feeder cells can be obtained from the patient or from healthy donor subjects.
  • IL-2 may be added to the REP culture at concentrations of about 6,000 U/rmL to promote rapid TIL cell division. Expansion of TILs in this manner can take about 2 weeks or longer, and results in a pool of about 10- 150 billion TILs.
  • the expanded cells may be washed and pooled, and may be suitable for infusion into a patient. Patients may typically receive 1 or 2 infusions (separated by 1 -2 weeks) of 10 9 ⁇ 10 11 cells.
  • IL-2 therapy e.g., 7.2x10 5 lU/kg every 8 hours for about 2 to about 3 days
  • IL-2 therapy e.g., 7.2x10 5 lU/kg every 8 hours for about 2 to about 3 days
  • a patient can optionally be lymphodepleted using cyclophosphamide (Cy) and fludaribine (Flu).
  • Cy cyclophosphamide
  • Flu fludaribine
  • TBI total body irradiation
  • Infusion of minimally expanded TIL to subjects receiving an ACT regimen may promote the persistence of the transferred cells, stimulate the persistence, proliferation and survival of transferred cells, and improve tumor regression.
  • PBMC peripheral blood mononuclear cells
  • antigen-specific T cells for ACT may be generated using multiple stimulation of autologous PBMC using artificial antigen-presenting cells (aAPC) expressing HLA-A0201 , costimulatory molecules, and membrane-bound cytokines.
  • aAPC artificial antigen-presenting cells
  • T cells can be rapidly expanded by stimulation of peripheral blood mononuclear cells (PBMC) in vitro with one or more antigens (including antigenic portions thereof, such as epitope(s), or a cell) of the cancer, which can be optionally expressed from a vector, in the presence of a T cell growth factor, such as 300 lU/ml IL-2 or IL-15, with IL-2 being preferred.
  • PBMC peripheral blood mononuclear cells
  • antigens including antigenic portions thereof, such as epitope(s), or a cell
  • T cell growth factor such as 300 lU/ml IL-2 or IL-15, with IL-2 being preferred.
  • the in wfro-induced T-cells may be rapidly expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells.
  • the T-cells can be re-stimulated with irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2, for example.
  • cell population may be enriched for CD8+ T cells.
  • a T cell culture may be depleted of CD4+ cells and enriched for CD8+ cells using, for example, a CD8 microbead separation (e.g., using a Clini-MACSPplus CD8 microbead system
  • CD8+ T cells may improve the outcome of ACT by removing CD4+ T regulatory cells.
  • Infusion of minimally expanded T cells, e.g., CD8+ and/or CD4+ T cells obtained from stimulation of PBMCs, to subjects receiving an ACT regimen may promote the persistence of the transferred cells, stimulate the persistence, proliferation and survival of transferred cells, and improve tumor regression.
  • minimally expanded T cells e.g., CD8+ and/or CD4+ T cells obtained from stimulation of PBMCs
  • T Cells Genetically Modified to Express a T Cell Receptor (TCR) that Specifically Recognizes a Tumor Antigen
  • TILs with high avidity for tumor antigens it may not be possible to obtain TILs with high avidity for tumor antigens in the quantity necessary for ACT. Accordingly, it may be desirable to genetically modify lymphocytes to obtain a cell population that may specifically recognize an antigen of interest prior to infusion into a subject.
  • Genes encoding TCRs can be isolated from T cells that specifically recognize cancer antigens with high avidity.
  • T lymphocytes isolated from peripheral blood can be transduced with a retrovirus or a lentivirus that contains genes encoding TCRs possessing the desired specificity. This method may permit the rapid production to a large number of tumor-antigen-specific T cells for ACT.
  • T cells may be transduced to express a T cell receptor (TCR) having antigenic specificity for a cancer antigen using transduction techniques described in Heemskerk et al. Hum Gene Ther. 19:496-510 (2008) and Johnson et al. Blood 1 14:535-46 (2009).
  • TCR T cell receptor
  • the content of these references is hereby incorporated by reference in their entireties.
  • ACT using T cells genetically modified to express a TCR recognizing an antigen of interest can be performed in accordance with the clinical trial protocol published by Morgan et al., Science (2006) 314(5796):126-129. The content of this reference is hereby incorporated by reference in its entirety.
  • T cells e.g., T cells that have been genetically engineered to express a TCR (or modified TCR) recognizing a tumor antigen
  • ACT regimen may promote the persistence of the transferred cells, stimulate the persistence, proliferation and survival of transferred cells, and improve tumor regression.
  • TAA peptides that are capable of use with the methods and embodiments described herein include, for example, those TAA peptides described in U.S. Publication 20160187351 , U.S. Publication 20170165335, U.S. Publication 20170035807, U.S. Publication 20160280759, U.S. Publication 20160287687, U.S. Publication
  • T cells described herein selectively recognize cells which present a TAA peptide described in one of more of the patents and publications described above.
  • T cell receptors capable of use with methods described herein, include, for example, those described in U.S. Publication No. 20170267738, U.S.
  • TAA that are capable of use with the methods and
  • T cells selectively recognize cells which present a TAA peptide described in SEQ ID NO: 1 - 157 or any of the patents or applications described herein.
  • T cells Genetic engineering of T cells to express a TCR having a desired specificity as described above may be a very promising approach for ACT. Notwithstanding, there is the potential for mispairing of the engineered TCR alpha and beta chains with endogenous TCR chains. In addition, the success of ACT using cells expressing engineered TCR depends on expression of the specific MHC molecule recognized by the TCR in the targeted cancer cells. To avoid these potential complications, T cells may alternatively be engineered to express chimeric antigen receptors (CARs).
  • CARs chimeric antigen receptors
  • CARs may contain an antigen binding domain coupled with the transmembrane domain and the signaling domain from the cytoplasmic tail of the CD3 z chain. There is some evidence that the CD3 z chain may be insufficient to fully activate transduced T cells. Accordingly, CARs may preferably contain an antigen binding domain, a costimulatory domain, and a CD3 z signaling domain. Using a costimulatory domain in combination with the CD3 z signaling domain mimics the two-signal model of T cell activation.
  • the CAR antigen binding domain can be an antibody or antibody fragment, such as a Fab or an scFv.
  • the antigen binding domain is separated from the CD3 z signaling domain and the costimulatory domain by a transmembrane domain.
  • the transmembrane domain may be derived from any transmembrane protein.
  • a transmembrane domain naturally associated with one of the domains in the CAR may be used.
  • an exogenous or synthetic transmembrane domain is used.
  • the transmembrane domain can be selected or modified by amino acid substitution to minimize interactions with other membrane proteins.
  • a spacer may optionally be incorporated.
  • the spacer may be any oligo- or polypeptide that functions to link the transmembrane domain to either the extracellular domain or the cytoplasmic domain.
  • a spacer may contain up to 300 amino acids, preferably 10 to 100 amino acids, and more preferably 25 to 50 amino acids.
  • the intracellular domain of a CAR may be responsible for activation of at least one of the normal effector functions of the immune cell, in which the CAR is expressed. Effector functions may include, for example, cytolytic activity or helper activity, such as the secretion of cytokines.
  • intracellular signaling domain of a molecule may refer to the portion of a protein, which transduces the effector function signal and directs the cell to perform a specialized function. While the entire intracellular signaling domain can be used, in many cases a portion of the intracellular domain may be used, so long as the selected portion transduces the effector function signal.
  • the cytoplasmic domain of a CAR can include the CD3 z signaling domain on its own, or in combination with a costimulatory domain.
  • the costimulatory domain contains the intracellular domain of a costimulatory molecule.
  • Costimulatory molecules may be cell surface molecules that promote an efficient response of lymphocytes to antigen.
  • the costimulatory domain may contain an intracellular domain of a costimulatory molecule, such as 4-1 BB, CD27, CD28, 0X40, CD30, CD40, PD-1 , ICOS, lymphocyte function-associated antigen-1 (LFA-1 ), CD2, CD7, LIGHT, NKG2C, B7-H3, a CD83 ligand, or combinations thereof.
  • the costimulatory molecule may be the intracellular domain of 4-1 BB or CD28.
  • Infusion of minimally expanded T cells e.g., T cells that have been genetically engineered to express a CAR recognizing a tumor antigen, to subjects receiving an ACT regimen may promote the persistence of the transferred cells, stimulate the persistence, proliferation and survival of transferred cells, and improve tumor regression.
  • T cells may be tested in non-cognate antigen or low cognate antigen environments.
  • manufactured T cells may be seeded in the culture at low densities, e.g., from about 1 ,000 to about 1 x10 6 cells/cm 2 , from about 1 ,000 to about 500,000 cells/cm 2 , from about 1 ,000 to about 250,000 cells/cm 2 , from about 1 ,000 to about 200,000 cells/cm 2 , from about 1 ,000 to about 150,000 cells/cm 2 , from about 1 ,000 to about 100,000 cells/cm 2 , from about 1 ,000 to about 50,000 cells/cm 2 , from about 1 ,000 to about 10,000 cells/cm 2 , or from about 1 ,000 to about 5,000 cells/cm 2 , in the absence of cognate antigen-presenting cells, e.g., cognate antigen-presenting tumor cells, dendritic cells, or macrophages.
  • cognate antigen-presenting cells e.g., cognate antigen-presenting tumor cells, dendritic cells, or macrophages.
  • manufactured T cells may be cultured in the non-cognate antigen or low cognate antigen environments in the presence of low concentrations of cytokines, e.g., from about 1 to about 1 ,000 ng/ml, from about 1 to about 500 ng/ml, from about 1 to about 250 ng/ml, from about 1 to about 100 ng/ml, from about 1 to about 50 ng/ml, from about 5 to about 50 ng/ml, from about 5 to about 40 ng/ml, from about 5 to about 30 ng/ml, from about 5 to about 20 ng/ml, or from about 5 to about 10 ng/ml, for a long period of time, e.g., from about 1 to about 30 days, from about 2 to about 25 days, from about 3 to about 21 days, from about 3 to about 14 days, from about 3 to about 10 days, or from about 3 to about 7 days.
  • cytokines e.g., from about 1 to about 1 ,000 ng/
  • T-cells were manufactured for 4, 7, or 10 days. After this manufacturing, the T-cells were analyzed via the CSA and the following metrics were analyzed: (1 ) cell survival as measured by fold growth of T-cells, (2) apoptosis as measured via propidium iodide and Annexin-V stain, (3) division as measured by the dilution of proliferation dye PkH67, (4) cytokine receptor expression as measured by flow cytometry, and (5) T-cell memory phenotype as measured by flow cytometry.
  • CSA shows prolonged expansions may lead to a significant reduction in the fitness of the T-cells when assessed within the CSA as assessed by the following observations: (1 ) decreased T-cell survival, (2) increased apoptosis, (3) decreased division rate, (4) cytokine receptor expression correlation, and (5) decreased survival of the Tnaive/scm compartment.
  • CSA was performed for 21 days, each sample was analyzed at 7 time points, which may define a single metric for temporal behavior.
  • the area under the curve (integration) of the temporal data was calculated and is used as a single defining metric to represent the behavior of the sample over the 21 days in the following results.
  • Apheresed T cells may be obtained from healthy allogenic donors or patients. These T cells may be activated or stimulated with activating anti-CD3 antibody, e.g., OKT3, in the presence of IL-2, or with anti-CD3- and anti-CD28 antibodies-coated paramagnetic beads in the presence of IL-2, or with artificial antigen presenting cell (aAPC) expressing 4- 1 BBL and an Fc receptor with OKT3 and IL-2. Activated T cells may then be transduced with recombinant TCR using retro- or lentiviral platform.
  • activating anti-CD3 antibody e.g., OKT3
  • aAPC artificial antigen presenting cell
  • Transduced T cells may be expanded for different lengths of time, for example, 4 days (Day 4), 7 days (Day 7), or 10 days (Day 10), in which activation starts on Day 0. Because recombinant TCR may be integrated into T cell genomes, all daughter cells generated during expansion may also express recombinant TCR. Expanded/transduced T cells may be used immediately or may be cryopreserved for future use.
  • FIG. 4 shows an embodiment of a cytokine sensitivity assay described herein.
  • cryopreserved or frozen expanded TCR-transduced T cells e.g., for 4 days, 7 days, or 10 days
  • Proliferation dye e.g., PkFI26 stain and respective cytokines (e.g., IL-2, IL-15, IL-7, or a combination thereof) at varying concentrations may be added and incubated for a period of time, for example, 21 days.
  • Fresh cytokines may be fed to cultured T cells every 7 days, i.e., on Day 0, Day 7, and Day 14, during the 21 -day assay. Towards the end of every 7 days in assay, culture media would have reduced levels of cytokines as compared with that at the start of assay.
  • expanded engineered T cells may be collected and analyzed for cell numbers, proliferation, apoptosis, e.g., via Annexin-V staining, memory phenotypes, e.g., CD45RO and CCR7 markers, and cytokine receptor expression, e.g., IL-2 receptor (CD25), IL-7 receptor (CD127), and IL-15 receptor (CD122).
  • FIGS. 5A-5D show phenotypes of TCR-transduced T cells, which were obtained from a healthy donor and expanded for (A) 0 day, (B) 4 days, (C) 7 days, and (D) 10 days. Expanded T cells were separated from lymphocytes by CD45RO staining and subsequently by CCR7 staining to discriminate Tnaive/Tscm (CD45RO-CCR7+), e.g., 23.2% (Day 4 expanded T cells), 16.4% (Day 7 expanded T cells), and 22.9% (Day 10 expanded T cells). Compared with Day 0 (49.4%, without expansion), Day 4, Day 7, and Day 10 expanded T cells show decreased number of cells with T SCm -like phenotype.
  • T cells expanded for 4 days, 7 days, or 10 days were cultured in the presence of IL-15 for 21 days.
  • Fresh IL-15 (10 ng/ml) was fed to cultured T cells every 7 days, i.e., on Day 0, Day 7, and Day 14, during the 21 -day assay.
  • T SCm -like phenotype was examined by flow cytometry using CD45RO and CCR7 staining at the end of every 7-day IL-15 feed, i.e., on Day 7, Day
  • FIGS. 6A-6I indicate that Day 4 expanded T cells exhibit better IL-15 sensitivity by retaining T SC m-like, i.e., Tnaive/Tscm, cell population throughout the 21 -day assay.
  • Tscm-like phenotypes correlate with T cell persistence in vivo, these results suggest that earlier expanded (for example, about 4 days) engineered T cells may be better than those expanded for a longer period of time, for example, greater or equal to about 7 days.
  • FIG. 20A shows, at Day 21 of the expansion, significantly higher percentages of na ' ive (scm) and central memory (T C m) T cells in the 3-day (early) expanded samples, while both of these less-differentiated T-cell compartments were drastically reduced in the 7-day (Mid) and 10-day (Late) expanded samples.
  • scm na ' ive
  • T C m central memory
  • FIG. 20B shows, consistently, there was an increase in proliferation of the CCR7- expressing cells based on PkH dilution by day 7 in the culturing period with IL-15, suggesting that reduced expansion may result in retention of proliferation potential through increased expression of cytokine receptors.
  • FIGS. 7A-7C show that T cells expanded at Day 4 exhibit higher cell growth or more surviving cells in the presence of (A) IL-7, (B) IL- 15, and (C) IL-2 over a 21 -day period as compared to those expanded for a longer period of time, for example, Day 7 and Day 10 expansion.
  • the dotted line is set at 1 to indicate no difference in fold growth relative to the starting number of cells.
  • cytokines for example, IL-2 (300 U/ml) (FIG. 8A), IL-7 (10.0 ng/ml) (FIG. 8B), or IL-15 (10.0 ng/ml) (FIG. 8C).
  • the integrated survival of each fold growth curve were determined by calculating the area under the curve. From an analysis of three biological donors, there was a trend in which the earlier expanded T cells outperformed the later expanded cells.
  • IL-2 there was an approximately 5-fold drop in survival between day 4 and day 7 expanded cells, with an approximately 2-fold drop in survival between day 7 and day 10 expanded cells.
  • IL-7 there was an approximately 6-fold drop in integrated survival between day 4 and day 7 expanded cells, with an approximately 4-fold drop between day 7 and day 10 expanded cells.
  • FIGS. 10A-10C show that T cells expanded for 4 days contain fewer apoptotic cells in the presence of (A) IL-7 (10 ng/ml), (B) IL-15 (10 ng/ml), and (C) IL-2 (300 IU/ml) as compared to those expanded for 7 and 10 days.
  • the % apoptosis of lymphocyte was gated by excluding debris and low FSC populations.
  • 1 1 A-1 1 C show, on Day 10 in the assay, lower integrated apoptosis, as determined by the area under curve, of TCR- transduced T cells expanded at about 4 days.
  • T cells expanded at 4 days contain fewer (4.97%, Annexin-V+/PI-)
  • A apoptotic cells than those expanded for a longer period of time, for example, (B) Day 7 (10.6%, Annexin-V+/PI-) and (C) Day 10 (18.2%, Annexin-V+/PI-), in the presence of IL-15 (10 ng/ml).
  • FIGS. 13A-13C show earlier expanded, e.g., Day 4, TCR-transduced T cells contain more dividing cells in the presence of (A) IL-7 (10 ng/ml), (B) IL-15 (10 ng/ml), and (C) IL-2 (300 IU/ml), as compared to those expanded for longer period of time, e.g., Day 7 and Day 10. Data is shown up to 10 days due to lack of cells in Day 10 cells after 10 days in assay.
  • cytokines e.g., IL-2 (300 U/ml) (FIG. 14A), IL-7 (10.0 ng/ml) (FIG. 14B), or IL-15 (10.0 ng/ml) (FIG. 14C).
  • the earlier expanded cells e.g., Day 4, underwent division as calculated by the percentage of cells which diluted the proliferation dye at each time point across 10 days in the CSA.
  • Cytokine sensitivity may be determined by the levels of cell division induced by cytokines. To determine cytokine sensitivity of expanded T cells, integrated cell division of expanded T cells induced by IL-2, IL-7, or IL-15 was measured in cytokine non-limiting conditions, e.g., 3 days in assay. Integrated cell division may be calculated by performing an integration by calculating the area under the curve of the cell division over 3 days in assay. FIGS.
  • T cells contain more dividing cells in the presence of (A) IL-7 (10.0 ng/ml), (B) IL-15 (10.0 ng/ml), and (C) IL-2 (300 IU/ml) as compared to those expanded for longer period of times, e.g., Day 7 and Day 10.
  • IL-7 10.0 ng/ml
  • B IL-15
  • C IL-2
  • results show shortened in vitro expansion of T cells respond to cytokines better than longer expanded T cells.
  • IL-2 300 U/ml
  • IL-7 (10.0 ng/ml
  • FIG. 16B shows IL-15 (10.0 ng/ml)
  • Cytokine sensitivity may also be determined by the expression levels of cytokine receptors that mediate cellular signaling pathways in the presence of cytokines.
  • CSA measures the response to cytokine induced survival, proliferation, and apoptosis. These changes may correlate with the expression of the respective cytokine receptors within each T-cell population at the beginning of the assay.
  • the expression of the defining subunit of the IL-2, IL-7, and IL-15 cytokine receptors i.e., CD25, CD127, and CD122, respectively, were measured.
  • CD122 is a shared subunit between the IL-2 and IL-15 receptors, though it is commonly assigned to be the reactive subunit of the IL-15 receptor.
  • FIG. 16D shows Day 4 expanded T cells, after 3 days in assay, express more IL- 2 receptor (CD25) as compared to those expanded for a longer period of time, e.g., Day 7 and Day 10.
  • CD122 IL-15 receptor
  • CD127 IL-7 receptor
  • results from these assays show that earlier manufactured (or minimally expanded) engineered T cells, for example about 3 to about 5 days, perform better as compared to longer expanded cells, for example, about 7 to about 10 days.
  • minimally expanded, for example about 3 to about 5 days engineered T cells may show greater clinical efficacy than that expanded for extended periods for example about 7 to about 10 days, in vitro, due to increased naivety, e.g., increased population of na ' ive T cells (TN) and/or stem memory T cells (T S cm)/T central memory (T C m), increased ability to proliferate, and increased persistence via, for example, decreasing apoptosis induced by CWID.
  • TN na ' ive T cells
  • T S cm stem memory T cells
  • T C m T central memory
  • EXAMPLE 7 [00190] Mechanism of Action (MOA) Phenotyping of Cells During CD3/CD28 Manufacturing
  • telomere length is a hallmark of dysfunctional cells as they become highly differentiated and eventually senescent.
  • fluorescence in situ hybridization assay was used to assess the relative telomere length (RTL) of the T-cells against an internal cell line control.
  • FIG. 22 shows, for all four donors (D1 -D4) analyzed, there was a loss in RTL throughout the expansion protocol, with the day 4 expansion cells having the highest RTL. There was an approximate 20% loss in RTL between day 4 and day 7 expanded cells and an additional 10% loss in RTL between day 7 and day 10 expanded cells when all donors were grouped together. There were signs of an age bias in the data as well, with the younger donors, on average, having longer RTL compared to the older donors when compared at the day 10 expanded time-point. Donors’ age: D1 : 50 years old, D2: 31 years old, D3: 49 years old, and D4: 45 years old.
  • the CSA results show there may be a distinct difference in the starting memory compartments between the differentially expanded cells.
  • a higher resolution analysis was performed on the starting memory compartment to detect the differences between the differentially expanded samples.
  • FIG. 24 shows there was a small statistically insignificant difference in the T naive/scm compartment between day 4, 7, and 10 (mean values of 20.03%, 1 1.1 %, 17.47 % of CD8 cells). There was, however, a statistically significant difference (p ⁇ 0.05) within the Tern compartment between day 4, 7, and 10 expanded cells (mean values 58.27, 37.73, 16.8 % of CD8 cells). There was a statistically significant difference (p ⁇ 0.05) within the Tem compartment between day 4, 7, and 10 expanded cells (mean values of 18.9, 48.13, and 58.9 % of CD8 cells).
  • RNA sequencing was performed from three biological donors expanded for 4, 7, or 10 days.
  • FIG. 26 shows, based on cluster analysis, distinct grouping of the day 4 expanded cells compared to the day 7, which appeared in an intermediary cluster, while the day 10 cells appeared in a unique cluster of their own.
  • RNA sequencing was analyzed for differentially expressed genes (DEGs) between the day 4, 7, and 10 expanded cells across three biological donors.
  • DEGs differentially expressed genes
  • FIG. 27 shows the gene expression profiles changed in the most early in the manufacturing process as evident by the 5,078 DEGs in the day 4 vs day 7 comparison and the 5,643 DEGs in the day 4 vs day 10 comparison.
  • the 5,078 DEGs in the day 4 vs day 7 comparison there was a roughly equal distribution of up and down regulated genes.
  • KEGG Kyoto Encyclopedia of Genes and Genomes
  • KEGG pathway analysis was performed to identify gene pathways that were over-represented in the differing gene sets.
  • KEGG pathways may be related to T- cell proliferation and persistence based on the functionality results obtained from the CSA, e.g., survival, division, and apoptosis.
  • FIG. 28 shows, comparing with the later time points to day 4 in manufacturing, there is a significant down regulation in DNA replication and cell cycle gene pathways. Compounding this effect, there was a significant up regulation in apoptosis, p53 signalling gene pathways during the same period in manufacturing. In agreement with the gene expression results in FIG. 27, there were very few significantly enriched pathways between day 7 and day 10 in manufacturing.
  • PBMCs Healthy donor whole blood was purchased from Flemacare and PBMCs were isolated by Ficoll gradient. PBMCs were activated for 16-24 hours in TexMACS (Miltenyi 130-097-196) supplemented with 5% Fluman AB serum (Gemini 100-318) media by plating at 1 x 10 6 live PBMC/ml on tissue culture flasks coated overnight with 1 ug/ml anti-CD3 (eBioscience 16-0037-85) and 1 ug/ml anti-CD28 (eBioscience 16-0289-85) antibody in PBS (Lonza 17-516F) at 4 degrees Celsius.
  • Cells were grown for an additional 2, 5, or 8 days depending on the desired manufacturing time (4, 7, or 10 total days). After manufacturing, cells were counted and frozen down at 5 x 10 6 /ml in Cyrostorel O, placed at -80 degrees Celsius for 16-24 hours and then stored long-term at LN2 vapor phase until needed.
  • Cell division may be measured by the dilution of proliferation dye PkH67.
  • PkH67 (Sigma PKH67GL) stain was performed per manufacturer’s protocol with the exception that the day 4 manufactured cells were stained at a 2X concentration to account for the larger cell size compared to day 7 or day 10 manufactured cells. PkH staining was performed before the flow cytometry viability dye stain.
  • T-cell products were thawed and rested for approximately four hours in
  • TexMACS supplemented with 5% Human AB serum and 100 U/mL Benzonase (Sigma E101 14) at 1 -2 x 10 6 /ml.
  • cells were labeled with PkH and 2 x 10 5 lymphocytes were cultured in a Grex24 well flask with a titration of IL-7, IL-15, or IL-2 (R&D Systems 202-IL) for a total of 21 days.
  • IL-7 IL-7
  • IL-15 IL-15
  • IL-2 R&D Systems 202-IL
  • Live cells were quantified and resuspended to 1 -2 x 10 6 live-cell/ml in PBS then stained with Live-Dead stain according to manufacturer’s protocol. Cells were then washed with Flow buffer and then resuspended at desired antibody concentrations as indicated in the tables below and stained for 15-30 minutes in the dark at 4 degrees Celsius, with the exception that the CCR7 stain was done at 37 degrees Celsius in RPMI (Gibco 1 1835-030) without serum. Cells were then washed in Flow buffer and resuspended in fixation buffer and stored at 4 degrees Celsius until acquired on the BD Fortessa or Miltenyi MACSQuant analyzer. The following tables contain the reagents used for all flow cytometry straining. [00223] Telomere length determination
  • T-cells were mixed at a 1 :1 ratio with control 1301 tumor cells (4N genome). Cells were then permeabilized and a Telomere PNA FITC probe was hybridized overnight. The next day, a counter propidium iodide stain was performed to discriminate intact cells and the cells were acquired by flow cytometry. The telomere length of the test cells was calculated as a ratio to that of the control 1301 tumor cell line.
  • Clonality was defined as 1 - Peilou’s eveness and was calculated on productive
  • rearrangement i and N is the total number of rearrangements.
  • Clonality values range from 0 to 1 and describe the shape of the frequency distribution: clonality values approaching 0 indicate a very even distribution of frequencies, whereas values approaching 1 indicate an increasingly asymmetric distribution in which a few clones are present at high frequencies.
  • Statistical analysis was performed in R version 3.2.
  • Downstream analysis was performed using a combination of programs including STAR, HTseq, Cufflink and our wrapped scripts. Alignments were parsed using Tophat program and differential expressions were determined through DESeq2/edgeR. GO and KEGG enrichment were implemented by the ClusterProfiler. Gene fusion and difference of alternative splicing event were detected by Star-fusion and rMATS software.
  • RNAseq (Novogene) Reads mapping to the reference genome
  • Reference genome and gene model annotation files were downloaded from genome website browser (NCBI/UCSC/Ensembl) directly. Indexes of the reference genome was built using STAR and paired-end clean reads were aligned to the reference genome using STAR (v2.5). STAR used the method of Maximal Mappable Prefix(MMP), which can generate a precise mapping result for junction reads.
  • MMP Maximal Mappable Prefix
  • RNAseq Novogene Quantification of gene expression level
  • HTSeq vO.6.1 was used to count the read numbers mapped of each gene. FPKM of each gene was then calculated based on the length of the gene and reads count mapped to this gene. FPKM, Reads Per Kilobase of exon model per Million mapped reads, accounts for the effect of sequencing depth and gene length for the reads count at the same time, and is commonly used method for estimating gene expression levels.
  • DESeq2 differential expression analysis between two conditions/groups (two biological replicates per condition) was performed using the DESeq2 R package (2_1.6.3).
  • DESeq2 provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting p-values were adjusted using the Benjamini and
  • Flochberg s approach for controlling the False Discovery Rate (FDR). Genes with an adjusted p-value ⁇ 0.05 found by DESeq2 were assigned as differentially expressed.
  • RNAseq Novogene
  • KEGG KEGG enrichment analysis of differentially expressed genes
  • Advantages of the present disclosure may include cytokine sensitivity assays that may be used to determine which types of in vitro manufactured T cells that may potentially persist in vivo by increasing proliferation and survival and decreasing apoptosis of transferred cells in a high-throughput patient specific fashion, thus, improve tumor regression and increase efficacy of ACT.

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Abstract

La présente invention concerne des procédés d'amélioration de l'efficacité de lymphocytes T. Selon un aspect, l'invention concerne en outre des procédés d'amélioration de la persistance de lymphocytes T pour un transfert cellulaire adoptif ou une thérapie cellulaire adoptive (ACT). L'invention concerne en outre des essais de sensibilité à la cytokine (CSA) et une méthodologie associée permettant de prédire la persistance de lymphocytes T perfusés adoptivement. L'invention concerne également des procédés de traitement du cancer chez un sujet en ayant besoin, ainsi que des populations de lymphocytes T produites par les procédés décrits ici.
PCT/US2019/023104 2018-03-21 2019-03-20 Procédés d'amélioration de la persistance de lymphocytes t perfusés adoptivement Ceased WO2019183181A1 (fr)

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CN201980027347.6A CN112512593A (zh) 2018-03-21 2019-03-20 增强过继输注t细胞持久性的方法
SG11202009082YA SG11202009082YA (en) 2018-03-21 2019-03-20 Methods of enhancing persistence of adoptively infused t cells
AU2019240039A AU2019240039A1 (en) 2018-03-21 2019-03-20 Methods of enhancing persistence of adoptively infused T cells
JP2020549771A JP7344898B2 (ja) 2018-03-21 2019-03-20 養子注入されたｔ細胞の持続性を増強する方法
CA3094344A CA3094344A1 (fr) 2018-03-21 2019-03-20 Procedes d'amelioration de la persistance de lymphocytes t perfuses adoptivement
KR1020207030058A KR20200133370A (ko) 2018-03-21 2019-03-20 입양 주입된 t 세포의 지속성 강화 방법
EA202092243A EA202092243A1 (ru) 2018-03-21 2019-03-20 Способы усиления персистенции адоптивно инфузированных т-клеток
EP19772552.6A EP3768330B1 (fr) 2018-03-21 2019-03-20 Procédés d'amélioration de la persistance de lymphocytes t perfusés adoptivement

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DE102018108612.1A DE102018108612A1 (de) 2018-03-21 2018-04-11 Verfahren zur erhöhung der persistenz von adoptiv infundierten t-zellen

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110747166B (zh) * 2019-10-11 2021-07-09 厦门大学 一种外周血t细胞的体外扩增培养方法
WO2022040631A1 (fr) * 2020-08-21 2022-02-24 Immatics US, Inc. Procédés d'isolement de lymphocytes t sélectionnés cd8+
US12144827B2 (en) 2021-02-25 2024-11-19 Lyell Immunopharma, Inc. ROR1 targeting chimeric antigen receptor

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EP3768330A4 (fr) 2021-05-19
KR20200133370A (ko) 2020-11-27
JP2021518121A (ja) 2021-08-02
EP3768330B1 (fr) 2026-04-15
CN112512593A (zh) 2021-03-16
SG11202009082YA (en) 2020-10-29
EP3768330A1 (fr) 2021-01-27
TW202003050A (zh) 2020-01-16
US11905529B2 (en) 2024-02-20
US20190292520A1 (en) 2019-09-26
EA202092243A1 (ru) 2021-01-29
CA3094344A1 (fr) 2019-09-26
JP7344898B2 (ja) 2023-09-14
MA52138A (fr) 2021-01-27
DE102018108612A1 (de) 2019-09-26
AU2019240039A1 (en) 2020-11-12
US20210017493A1 (en) 2021-01-21

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